ABSTRACT (Core B: Viral Genetics Core) Viral rebound from latent HIV-1 reservoirs is the major threat for HIV-positive patients. One major hurdle in defining viral rebound is the inability of current technology to accurately quantify the latent HIV-1 viral pool and track the reactivation and viral expansion events that contribute to the rebound process. The overall goal of this program project is to develop a better understanding of HIV rebound using the humanized BLT (bone marrow- liver-thymus) mouse model for HIV infection and latency. To improve this model, we have developed a barcode approach to track the HIV infection during initial acute infection, ART suppression (latency) and rebound with Drs. Matt Marsden and Jerry Zack. We have also developed highly sensitive sequencing approach with Dr. Otto Yang to define viral quasi-species in HIV patient samples, which will be applied to our analysis of viral sequences in this project. In pursuit of the goals of the P01, Core B will provide novel viral genetic technology. The rationale for this technology is to enable more precise measurements of HIV-1 latently infected cells that are induced to express in vivo, and to more effectively monitor the viral strains that emerge and predominate in the plasma and tissues during rebound. Our innovative strategy involves barcoding individual HIV-1 viruses and then using them for tracking infection, thereby enabling the marking of each latently infected cell with a unique barcode identifier, and thereafter progeny viruses. The barcoded HIV-1 latent pool will then allow us to track the viral activity (transcription and virion production) in each latently infected cell as well as the fate of these latently infected cells by sequence analysis of the barcodes present in samples harvested from in vitro and in vivo experiments. The objective of this Viral Genetic Core is to provide technical support for tracking HIV during acute infection, latency and rebound with the following specific aims: Specific Aim 1. Construction and characterization of barcoded HIV virus libraries. Specific Aim 2. Sequencing-analyses the barcoded libraries during acute infection, latency and rebound. We will provide barcode technology to understand the tissue location of latent reservoirs, how tissue/cellular environment and immune responses impact reactivation potentials, and subsequent expansion/elimination of the viral pool by various treatment and immune responses.